The goal of this proposal is to develop new software for designing polymerase chain reaction (PCR) DNA amplifications in various formats based upon a rigorous scientific and computational approach. DNA Software Inc. has created the "oligonucleotide modeling platform," OMP, to enable robust automated high-throughput design of a variety of hybridization-based assays. The new software will significantly improve the reliability of PCR and will enable high-throughput PCR applications resulting in significant cost savings. Three specific aims will be performed: Aim1: OMP Core Development. An evolutionary algorithm will be added to allow optimization of the choice of primers for multiplexing, temperature cycling protocol, [Mg2+], strand concentration, and choice of enzyme. New functionality for simulating the kinetics of PCR hybridization and polymerase extension will be added to OMP. This module will allow for the time course of the reactions that occur during PCR cycling to be accurately modeled so that the product distribution at the end of the thermocycling is accurately predicted. Aim2: Develop a generalized graphical user interface (GUI) and internal functionality for PCR in different formats. The GUI will be configurable for many different PCR formats, and this proposal will focus on finishing the functionality required for multiplex PCR, gene synthesis, real-time PCR, and mismatched primer PCR. Aim3: Experimental studies for kinetics parameterization and validation of OMP for multiplex PCR and other formats. Fluorescence correlation spectroscopy and stopped-flow kinetics will be used to perform a systematic study of the kinetic constants for duplex formation, target unfolding, mismatch discrimination under different conditions, and several different sequences will be performed. To validate the predictions of our software for PCR primer design, we will perform a carefully designed series of single target and multiplex PCR reactions. [unreadable] [unreadable]